Metabolism and function of NK cells / 中国免疫学杂志

NK cells are important components of innate immune system and play a key role in immune responses.Activation of NK cells mainly depends on dynamic balance between activatory and inhibitory receptors expressed on surface.However,in many chronic diseases,balance between these receptors of NK cells is disorder,resulting in reduced cytolysis activity and cytokine produc-tion,which is in a state of immune inactivation.In recent years,many studies have shown that intracellular metabolism is crucial for immune cells such as NK cells,and changes of metabolism will regulate functions of immune cells by affecting cell development,pro-liferation and activity.In view of powerful anti-tumor and anti-viral effects of NK cells and their important clinical application value,it is important to study their metabolic characteristics and mechanisms.This review mainly introduces metabolic patterns of NK cell,related regulatory pathways,regulatory effects of metabolism on NK cell development,memory and functions,and metabolism-based NK cell therapy,also expounds important role of metabolism on NK cell biosynthesis,stability and effector function in vivo,as well as metabolism-related factors involved in NK cell inactivation in different chronic diseases,providing a solid research basis for clinical application of NK cell therapy.

Tongxie Yaofang Regulates Expression of NKG2DL to Enhance Anti-tumor Effect of NK Cells in Colon Cancer under Chronic Stress / 中国实验方剂学杂志

ObjectiveTo observe the effect of Tongxie Yaofang on the function of tumor-related natural killer （NK） cells under chronic stress and explore the possible molecular mechanism. MethodFifty SPF-grade BABL/C male mice were randomized into normal， model， and low-， medium-， and high-dose （6.825， 13.65， and 27.3 g·kg-1， respectively） Tongxie Yaofang groups， with 10 mice in each group. Other groups except the blank group were subjected to 7 days of chronic restraint stress， and then forced swimming and tail suspension tests were carried out to evaluate the modeling performance. After the successful modeling， rats in Tongxie Yaofang groups were administrated with low-， medium-， and high-doses of Tongxie Yaofang by gavage， while those in the other groups were administrated with normal saline by gavage. After 14 days， each group of mice was inoculated with subcutaneous colon cancer to establish the model of colon cancer under chronic stress. The pathological changes of the tumor tissue in each group of mice were observed using hematoxylin-eosin （HE） staining. The content of CD49b-positive cells in the peripheral blood and tumor tissue of mice was measured by flow cytometry. Enzyme-linked immunosorbent assay （ELISA） was employed to measure the content of molecules associated with NK cell activation in the peripheral blood. Western blot was employed to determine the protein levels of major histocompatibility complex class Ⅰ polypeptide-related sequences A and B （MICA+MICB） and UL-16-binding protein 1 （ULBP1） in the tumor tissue. ResultCompared with the normal group， the model group showed a decrease in 5-hydroxytryptamine （5-HT） content and an increase in corticosterone （CORT） content in the serum （P<0.05）. Compared with the model group， Tongxie Yaofang increased the 5-HT content and decreased the CORT content （P<0.05， P<0.01）. Compared with the normal group， the modeling increased the tumor volume and weight （P<0.05）， while Tongxie Yaofang inhibited such increases with no statistical significance. The tumor cells in the model group presented neat arrangement， irregular shape， uneven size， obvious atypia， common nuclear division， and small necrotic area， and blood vessels were abundant surrounding the tumor cells. Compared with the model group， Tongxie Yaofang groups showed sparse arrangement of tumor cells， different degrees of patchy necrosis areas in the tumor， and karyorrhexis， dissolution， and nuclear debris in the necrotic part. Compared with the normal group， the model group showed reduced CD49b-positive cells in the peripheral blood and tumor tissue （P<0.01）. Compared with the model group， Tongxie Yaofang increased CD49b-positive cells （medium dose P<0.01， high dose P<0.05， P<0.01）. Compared with the normal group， the modeling lowered the serum levels of granzymes-B （Gzms-B）， perforin （PF）， interferon （IFN）-γ， and tumor necrosis factor （TNF）-α （P<0.05， P<0.01）. Compared with the model group， low-dose Tongxie Yaofang elevated the serum levels of PF， Gzms-B， and TNF-α （P<0.05， P<0.01）， and medium-dose Tongxie Yaofang elevated the serum levels of Gzms-B， PF， IFN-γ， and TNF-α （P<0.05， P<0.01）. In addition， high-dose Tongxie Yaofang elevated the serum levels of PF， IFN-γ， and TNF-α （P<0.01）. Compared with the normal group， the model group presented down-regulated protein level of ULBP1 （P<0.05）. Compared with the model group， low-， medium-， and high-dose Tongxie Yaofang up-regulated the protein level of ULBP1 （P<0.05， P<0.01）， and medium- and high-dose Tongxie Yaofang up-regulated the protein level of MICA+MICB （P<0.05， P<0.01）. ConclusionTongxie Yaofang may promote NK cell activation by up-regulating the expression of MICA+MICB and ULBP1， thereby delaying the progression of colon cancer under chronic stress.

Anti-PD-L1 antibody enhances curative effect of cryoablation via antibody-dependent cell-mediated cytotoxicity mediating PD-L1highCD11b+ cells elimination in hepatocellular carcinoma

Cryoablation (CRA) and microwave ablation (MWA) are two main local treatments for hepatocellular carcinoma (HCC). However, which one is more curative and suitable for combining with immunotherapy is still controversial. Herein, CRA induced higher tumoral PD-L1 expression and more T cells infiltration, but less PD-L1highCD11b+ myeloid cells infiltration than MWA in HCC. Furthermore, CRA had better curative effect than MWA for anti-PD-L1 combination therapy in mouse models. Mechanistically, anti-PD-L1 antibody facilitated infiltration of CD8+ T cells by enhancing the secretion of CXCL9 from cDC1 cells after CRA therapy. On the other hand, anti-PD-L1 antibody promoted the infiltration of NK cells to eliminate PD-L1highCD11b+ myeloid cells by antibody-dependent cell-mediated cytotoxicity (ADCC) effect after CRA therapy. Both aspects relieved the immunosuppressive microenvironment after CRA therapy. Notably, the wild-type PD-L1 Avelumab (Bavencio), compared to the mutant PD-L1 atezolizumab (Tecentriq), was better at inducing the ADCC effect to target PD-L1highCD11b+ myeloid cells. Collectively, our study uncovered the novel insights that CRA showed superior curative effect than MWA in combining with anti-PD-L1 antibody by strengthening CTL/NK cell immune responses, which provided a strong rationale for combining CRA and PD-L1 blockade in the clinical treatment for HCC.

Research progress in leveraging biomaterials for enhancing NK cell immunotherapy / 浙江大学学报·医学版

NK cell immunotherapy is a promising antitumor therapeutic modality after the development of T cell immunotherapy. Structural modification of NK cells with biomaterials may provide a precise, efficient, and low-cost strategy to enhance NK cell immunotherapy. The biomaterial modification of NK cells can be divided into two strategies: surface engineering with biomaterials and intracellular modification. The surface engineering strategies include hydrophobic interaction of lipids, receptor-ligand interaction between membrane proteins, covalent binding to amino acid residues, click reaction and electrostatic interaction. The intracellular modification strategies are based on manipulation by nanotechnology using membranous materials from various sources of NK cells (such as exosome, vesicle and cytomembranes). Finally, the biomaterials-based strategies regulate the recruitment, recognition and cytotoxicity of NK cells in the solid tumor site in situ to boost the activity of NK cells in the tumor. This article reviews the recent research progress in enhancing NK cell therapy based on biomaterial modification, to provide a reference for further researches on engineering NK cell therapy with biomaterials.

Comparative Study of the Two High-Efficient Strategies for in vitro Generation of Human Umbilical Cord Blood-derived Natural Killer Cells / 中国实验血液学杂志

OBJECTIVE@#To explore the similarities and variations of biological phenotype and cytotoxicity of human umbilical cord blood natural killer cells (hUC- NK) after human umbilical cord blood-derived mononuclear cells (hUC-MNC) activated and expanded by two in vitro high-efficient strategies.@*METHODS@#Umbilical cord blood mononuclear cells (MNC) from healthy donor were enriched by Ficoll-based density gradient centrifugation. Then, the phenotype, subpopulations, cell viability and cytotoxicity of NK cells derived from Miltenyi medium (denoted as M-NK) and X-VIVO 15 (denoted as X-NK) were compared using a "3IL" strategy.@*RESULTS@#After a 14-day's culture, the contents of CD3-CD56+ NK cells were elevated from 4.25%±0.04% (d 0) to 71%±0.18% (M-NK) and 75.2%±1.1% (X-NK) respectively. Compared with X-NK group, the proportion of CD3+CD4+ T cells and CD3+CD56+ NKT cells in M-NK group decreased significantly. The percentages of CD16+, NKG2D+, NKp44+, CD25+ NK cells in X-NK group was higher than those in the M-NK group, while the total number of expanded NK cells in X-NK group was half of that in M-NK group. There were no significant differences between X-NK and M-NK groups in cell proliferation and cell cycle, except for the lower percentage of Annexin V+ apoptotic cells in M-NK group. Compared with X-NK group, the proportion of CD107a+ NK cells in M-NK group were higher under the same effector-target ratio (E∶T) (P<0.05).@*CONCLUSION@#The two strategies were adequate for high-efficient generation of NK cells with high level of activation in vitro, however, there are differences in biological phenotypes and tumor cytotoxicity.

Preliminary study on the effects of belimumab combined with classic therapy on peripheral blood NK cells in patients with systemic lupus erythematosus / 中华风湿病学杂志

Objective:To assess the changes of peripheral blood NK cells in patients with systemic lupus erythematosus (SLE) following belimumab and classic therapy.Methods:From January 2020 to March 2022, peripheral lymphocyte subsets were detected by flow cytometry in SLE patients treated with Belimumab and classic therapy. The duration of treatment was 24 weeks. A total of 40 treated SLE patients were enrolled. The lymphocyte subsets in healthy donors were used as normal control group. Paired sample t-test, rank-sum test, Spearman correlation and generalized linear mixed model were used for statistical analysis. Results:In contrast to healthy subjects, the numbers of NK cells in SLE patients before treatment were significantly decreased [276.0 (179.8, 384.0) cells/μl vs. 61.4 (43.0, 105.1) cells/μl; Z=-7.32, P<0.001], although that after treatment was higher than that before treatment [61.4 (43.0, 105.1) cells/μl vs. 107.7 (72.5, 186.5) cells/μl; Z= -3.22, P<0.001]. Generalized linear mixed model results showed that the increase in serum levels of C3 ( t= -2.94, P=0.006) and NK cells ( t=-2.25, P=0.031) were associated with a decrease of anti-dsDNA antibody titers. The cutoff value of elevated counts of NK cells after treatment was equal or more than 38.5 cells/μl with a sensitivity of 61.9% and a specificity of 81.2%. Compared with those with elevated counts of NK cells ≤38.5 cells/μl, patients with elevated counts of NK cells >38.5 cells/μl had a bigger difference anti-dsDNA antibody [49.2 (0.2, 207.2) vs. 156.2 (19.8, 260.7); Z=-1.55, P=0.120] and a bigger difference of SLEDAI-2000[4.5 (0.0, 10.0) vs. 13.0 (4.5, 18.0); Z=-2.52, P=0.012]. Conclusion:The change in the numbers of NK cells may serve as biomarkers for evaluating the therapeutic responses of SLE. Combinatory approaches employing belimumab and classic therapy may control SLE disease by increasing the number of NK cells

Atypical Lymphocytosis:Report of Two Cases and Literature Review / 中国医学科学院学报

To improve the diagnosis of atypical lymphocytes and reduce the misdiagnosis rate,we analyzed the medical records of 2 cases with cell morphology suggestive of atypical lymphocytes.One case was diagnosed with infectious mononucleosis and the other with aggressive NK cell leukemia.The purpose of this paper is to emphasize that the diagnosis of atypical lymphocytes based only on morphological interpretation of cells may be incorrect,which should be combined with clinical symptoms,signs,imaging examination,cell immunophenotype,and disease outcome.

The roles of immune cells in Behçet's disease

Abstract Behçet's disease (BD) is a systemic vasculitis that can affect multiple systems, including the skin, mucous membranes, joints, eyes, gastrointestinal and nervous. However, the pathogenesis of BD remains unclear, and it is believed that immune-inflammatory reactions play a crucial role in its development. Immune cells are a critical component of this process and contribute to the onset and progression of BD. By regulating the function of these immune cells, effective control over the occurrence and development of BD can be achieved, particularly with regards to monocyte activation and aggregation, macrophage differentiation and polarization, as well as T cell subset differentiation. This review provides a brief overview of immune cells and their role in regulating BD progression, which may serve as a theoretical foundation for preventing and treating this disease.

Cell phenotypes as activity biomarkers in patients with Systemic Lupus Erythematosus

Abstract The pathogenesis of systemic lupus erythematosus (SLE) is complex. Few studies in Brazilian population have addressed cell phenotypes associated with immunological responses and their associations with SLE activity. The aim of this study is to investigate cell phenotypes associated to SLE diagnosis, treatment and activity. Twenty-eight SLE female patients (17 inactive, 11 active) and 10 healthy women were included in this study. Markers of natural killer (Nk), T and B cells in peripheral blood were evaluated by flow cytometry. Nkt cells were decreased only in SLE active patients. Activated CD4+, regulatory T FoxP3+ and B cells were decreased in both active and inactive SLE patients, compared to control group. The data corroborate the disruption of immune regulatory response in SLE patients and suggest phenotipic changes as possible biomarkers of SLE activity.

Expression Level of SOCS3 in Acute Lymphoblastic Leukemia Cells Affects the Cytotoxicity of NK Cells / 中国实验血液学杂志

OBJECTIVE@#To detect the expression level of suppressors of cytokine signaling 3 （SOCS3） in acute lymphoblastic leukemia (ALL), and to observe the effect of over-expresson of SOCS3 in Jurkat cells on the cytotoxicity of NK cells.@*METHODS@#The expression levels of SOCS3 mRNA in peripheral blood mononuclear cells of 20 children with ALL and 20 healthy children (normal control group) were detected by RT-PCR. The peripheral blood NK cells from healthy subjects were selected by immunomagnetic technique, and the purity was detected by flow cytometry. SOCS3 was overexpressed in Jurkat cells infected with lentivirus vector, and SOCS3 mRNA expression was detected by RT-PCR after lentivirus infection. The NK cells were co-cultured with the infected Jurkat, and LDH release method was used to detect the cytotoxicity of NK cells on the infected Jurkat cells. The concentrations of TNF-α and IFN-γ were determined by ELISA. The expression of NKG2D ligands MICA and MICB on the surface of Jurkat cells were detected by flow cytometry. Western blot was used to detect the effect of SOCS3 overexpression on STAT3 phosphorylation in Jurkat cells.@*RESULTS@#Compared with the control group, the mRNA expression of SOCS3 in the peripheral blood mononucleated cells of ALL children was significantly decreased. The purity of NK cells isolated by flow cytometry could reach more than 70%. The expression of SOCS3 mRNA in Jurkat cells increased significantly after lentivirus infection. Overexpression of SOCS3 in Jurkat cells significantly promoted the killing ability of NK cells and up-regulated the secretion of TNF-α and IFN-γ from NK cells. The results of flow cytometry showed that the expression of NKG2D ligands MICA and MICB on Jurkat cells increased significantly after SOCS3 overexpression. Western blot results showed that overexpression of SOCS3 significantly reduced the phosphorylation level of STAT3 protein in Jurkat cells.@*CONCLUSION@#SOCS3 mRNA expression was significantly decreased in ALL patients, and overexpression of SOCS3 may up-regulate the expression of MICA and MICB of NKG2D ligands on Jurkat cell surface through negative regulation of JAK/STAT signaling pathway, thereby promoting the cytotoxic function of NK cells.

Comparison of the characteristics of NK cells after two different methods of expansion and observation of the clinical efficacy in patients who relapsed post allogeneic hematopoietic stem cell transplantation / 中华血液学杂志

Objective: To explore the differences in the biological effects of different expansion systems on natural killer (NK) cells, as well as the safety and preliminary clinical efficacy in the treatment of patients with recurrence after allogeneic hematopoietic stem cell transplantation (allo-HSCT) . Methods: Peripheral blood cells from healthy donors were stimulated with either CD3 combined with CD52 or K562 feeder cells loaded with IL-21/4-1BB to induce NK cell expansion. Changes in the NK cell phenotype, cytokine secretion, and cytotoxicity before and after expansion were detected. We also evaluated the safety and clinical efficacy of two different expansion strategies for patients received NK infusion. Results: Compared with the CD3/CD52 monoclonal antibody amplification system, the feeder cell expansion group had a higher purity of NK cells and higher expression ratios of NK cell surface activation receptors such as DNAM-1 and NKp30, while inhibitory receptor CTLA-4 expression was low and NKG2D/CD25/CD69/ Trail/PD-1/TIM-3/TIGIT had no statistically significant differences between the groups. Further functional results showed that the expression level of KI67 in NK cells after expansion in the two groups increased significantly, especially in the feeder cell expansion group. Simultaneously, the perforin and granzyme B levels of NK cells in the feeder cell expansion group were significantly higher than in the CD3/CD52 expansion group. A retrospective analysis of eight patients who received monoclonal antibody-expanded NK cell reinfusion and nine patients with trophoblast cell-expanded NK cell reinfusion was done. The disease characteristics of the two groups were comparable, NK cell reinfusion was safe, and there were no obvious adverse reactions. Clinical prognostic results showed that in the CD3/CD52 monoclonal antibody amplification group, the MRD conversion rate was 50% (2/4) , and the feeder cell expansion group was 50% (3/6) . After 5 years of follow-up from allo-HSCT, three patients in the monoclonal antibody expansion group had long-term survival without leukemia, and the remaining five patients had died; two patients died in the feeder cell expansion group, and the other six patients had long-term survival. Six cases had GVHD before NK cell reinfusion, and GVHD did not aggravate or even relieved after NK cell reinfusion. Conclusions: Preliminary results show that the biological characteristics of NK cells with diverse expansion strategies are significantly different, which may affect the clinical prognosis of patients with recurrence or persistent minimal residual disease after HSCT. The two groups of patients treated with NK cells from different expansion strategies had no obvious adverse reactions after NK cell infusion, but efficacy still needs to be further confirmed.

Expression of Killer Cell Immunoglobulin-like Receptors (KIR) in Sex-associated Malignancies

@#Introduction: Sex shapes immune response with possible consequence on tumor immune escape. Acute lymphoblastic leukemia (ALL) predominates in males while ovarian cancer (OC) occurs in females. NK cells essential for tumor killing may have male preponderance. Association of sex, NK cell activity and malignancies is unclear. We hypothesize that sex differentially affects KIR expressions in sex-biased cancers. Method: Expression of inhibitory (KIR2DL1-5 and KIR3DL1-3) and activating (KIR2DS1-2 and 4-5 and KIR3DS1) genes in B-, T-cell ALL, OC and normal controls were determined by reverse-transcription polymerase-chain-reaction. Result: All normal males (but not females) expressed the framework genes and generally maintained haplotype A, except KIR3DL1. Normal females expressed more activating KIRs. Frequencies of KIR2DL1, 2DL4 and 2DS2 were significantly reduced among ovarian cancer patients. Sex difference in frequencies of KIR expression was not detected in ALL as majority were undetectable except framework gene KIR3DL2, was more frequent among T-ALL. Conclusion: Cancers may be associated with reduced KIR expression and influence of sex requires investigation.

Xiaoai Jiedu Prescription-containing Serum Enhances Lethal Effect of NK Cells on Colon Cancer Cells / 中国实验方剂学杂志

ObjectiveTo explore the influence of Xiaoai Jiedu prescription （XJP）-containing serum on natural killer （NK） cells′ lethal effect on colon cancer cells and the molecular mechanism. MethodXJP-containing serum （0.1%， 0.5%， 1%， 5%， 10%） was used to treat HCT-116 cells and NK-92MI cells respectively for 24 h， and methyl thiazolyl tetrazolium （MTT） assay was employed to detect cell proliferation. Then， low-concentration （0.1%， 0.5%， 1%） XJP-containing serum was selected to treat co-cultured HCT-116 cells and NK-92MI cells for 24 h and calcein acetoxymethyl ester/propidium iodide （Calcein-AM/PI） was applied to detect the killing effect of NK cells on colon cancer cells. Flow cytometry was used to detect apoptosis of colon cancer cells， Western blot the expression of apoptosis-related proteins and signal transducer and activator of transcription 4 （STAT4） pathway-related proteins， and enzyme-linked immunosorbent assay （ELISA） the secretion of interferon （IFN）-γ. ResultHigh-concentration （5%， 10%） XJP-containing serum inhibited the proliferation of HCT-116 and NK-92MI cells （P<0.01）， while low-concentration （0.1%， 0.5%， 1%） XJP-containing serum had no obvious influence on cell proliferation compared with the blank group. As compared with the blank group， low-concentration XJP-containing serum enhanced the killing activity of NK cells against colon cancer cells in a concentration-dependent manner （P<0.01）， and induced apoptosis of colon cancer cells （P<0.01）. Moreover， XJP-containing serum （0.1%， 0.5%， 1%） down-regulated the expression of B-cell lymphoma 2 （Bcl-2） and B-cell lymphoma-extra large （Bcl-xl）， and up-regulated the expression of Bcl-2-associated X （Bax） compared with the blank group （P<0.05， P<0.01）. Compared with the co-culture group， XJP-containing serum （0.1%， 0.5%， 1%） increased the expression of p-STAT4 and IFN-γ （P<0.05）. ELISA result showed that XJP-containing serum （0.1%， 0.5%， 1%） raised IFN-γ secretion （P<0.01）. ConclusionXJP-containing serum can enhance the activity of NK cells to kill colon cancer cells. The mechanism is the likelihood that it activates STAT4 pathway， increases IFN-γ secretion by NK cells， down-regulates the expression of Bcl-xl and Bcl-2， and up-regulates the expression of Bax， thereby promoting the apoptosis of colon cancer cells.

Si Junzitang Regulates NKG2A Expression to Improve Anti-colon Cancer Function of NK Cells / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Si Junzitang in regulating the expression of NKG2A to affect the anti-colon cancer function of natural killer （NK） cells. MethodNK cells isolated from healthy honors were cultured and used to construct the three incubation models of NK cells， human colon cancer HCT116 cells， and NK cells + HCT116 cells （co-incubation）. real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was conducted to determine the mRNA levels of natural killer group 2 member A （NKG2A） and interleukin （IL）-15 in NK cells， as well as the mRNA level of histocompatibility leucocyte antigen E （HLA-E） in HCT116 cells. The secretion of IL-15 was detected by enzyme-linked immunosorbent assay （ELISA）. Methyl thiazolyl tetrazolium （MTT） assay was employed to determine the applicable concentration of IL-15 and test the effects of Si Junzitang and IL-15 on the activities of NK cells and the HCT116 cells in the co-incubation model. The effects of Si Junzitang and IL-15 on the mRNA levels of NKG2A in NK cells and HLA-E in HCT116 cells were detected by Real-time PCR. Monalizumab （M， anti-NKG2A mab） was used to block the NKG2A-HLA-E pathway in co-incubation model， and then the proliferation of HCT116 cells was detected by MTT assay. ResultThe interaction of NK cells and HCT116 cells up-regulated the mRNA levels of NKG2A in NK cells and HLA-E in HCT116 cells （P<0.05）， as well as the expression level and secretion of IL-15 （P<0.05）. Compared with the blank group， Si Junzitang and Si Junzitang + IL-15 promoted the proliferation and improved the anti-colon cancer function of NK cells （P<0.01）. Furthermore， they down-regulated the mRNA levels of NKG2A in NK cells and HLA-E in the HCT116 cells co-incubated with NK cells （P<0.01）. M and IL-15 + M inhibited the proliferation of HCT116 cells compared with the groups without M （P<0.01）. ConclusionThe interaction of NK cells and HCT116 cells can induce activation of NKG2A-HLA-E pathway to impair NK cell function. Si Junzitang can inhibit the activation of NKG2A-HLA-E pathway to restore the anti-colon cancer function of NK cells.

Recovery of IFN-7-secreting T and NK cells after low dose X-ray radiation injury and the protective effect of Prepared Radix Rehmanniae / 中国药理学通报

Aim To investigate the reeovery of I FN-7- tion and the protective effect of Prepared Radix Reh- secreting T and NK cells after low dose X-ray irradia- manniae ( PRR ).Methods X-ray 2.5Gy was used to establish a mouse irradiated model,and some irradiated mice were used to establish a melanoma lung cancer metastasis model or to be treated by PRR.The propor¬tion and number of Tel , Thl and NK1 cells with the phenotypes of IL-12R and IL-15R were detected by flow cytometry.The transcription levels of IL-12,1L- 15 ,STAT4 and T-bet were detected by RT-qPCR.Re¬sults Within four days after irradiation,Thl ,Tcl and NK1 cells were significantly reduced ( P < 0.05 , P < 0.01).The expression of IL-12R and IL-15R de¬creased in Thl and Tel cells , and increased in NK1 cells ( P < 0.05 ).On day 8 of irradiation , NK1 and Tc 1 cells recovered or exceeded basic level, but Th 1 was still lower than normal level ( P < 0.05 ) , and tumor load of irradiated mice increased significantly (P <0.01).Compared with radiation group, the propor¬tion and absolute numbers of NK1 ,Tel and Thl were up-regulated by PRR (P <0.05 <0.01) ,and tumor load was down-regulated ( P < 0.05 ).Conclusions The impaired reconstitution of Thl cells after irradia¬tion affects the anti-tumor ability.PRR promotes the recover}' of IFN-∗y-secreting T and NK cells after radia¬tion and reduces the risk of tumor metastasis in mice.

Study on effect of restoring mitochondrial dynamics on NK2 activation of NK cells / 中国药理学通报

Aim To explore the internal mechanism of NK2 activation of NK cells from the perspective of "mitochondrial dysfunction-abnormal cell activation".Methods NK-92MI cells were divided into blank group, TSLP group, 1, 5, and 10 μmol·L-1 Mdivi-1 dose groups.The levels of IL-4, IL-5, and IFN-γ in the supernatant of each group were determined by ELISA; The expression of p-Drp1 and MnSOD protein in each group was determined by Western blotting; the ROS level of each group was detected by DHE staining and flow cytometry; mitochondrial morphology was observed by confocal laser in each group of cells.Results ELISA showed that compared with control group, the levels of IL-4 and IL-5 in cell supernatant of TSLP group significantly increased, and the level of IFN-γ was down-regulated(P<0.05); Compared with TSLP group, the levels of IL-4 and IL-5 in cell supernatant of 5 and 10 μmol·L-1 Mdivi-1 group decreased, and the IFN-γ concentration of the 10 μmol·L-1 Mdivi-1 group rose(P<0.05).DHE staining and flow cytometry showed that ROS level of cells in TSLP group was significantly higher than control group.Compared with TSLP group, ROS level of the 5 and 10 μmol·L-1 Mdivi-1 groups decreased(P<0.05).The laser confocal results showed that after TSLP stimulation, a large number of spherical mitochondria were formed in cells.This phenomenon was improved to a certain extent after the intervention of 5, 10 μmol·L-1 Mdivi-1.Western blot analysis showed that the p-Drp1 level of NK-92MI cells in TSLP group was significantly up-regulated, and the expression of MnSOD decreased, while the intervention of Mdivi-1 effectively reversed the changes in the expression of the above-mentioned molecules.Conclusions Mitochondrial dynamic imbalance may be one of the internal mechanisms of abnormal activation of NK cells, and it may be an important target for regulating NK2 activation of NK cells and improving the allergic inflammatory response mediated by it.

Células asesinas naturales con el receptor de antígeno quimérico (CAR-NK): terapia emergente contra el cáncer / Chimeric antigen receptor-natural killer cells: novel therapy against cancer

Resumen Una de las herramientas más novedosas en inmunoterapias adoptivas contra leucemias y tumores malignos es el uso del receptor de antígeno quimérico "CAR". El receptor CAR ha sido ampliamente utilizada en células T (células CAR-T) potenciando su eficacia en el reconocimiento y eliminación de tumores, obteniéndose a la fecha terapias basadas en esta tecnología. No obstante, las células CAR-T llegan a repercutir negativamente en la salud del paciente, presentando el síndrome neurológico de efecto inmune asociado a células (ICANS) y el síndrome de lanzamiento de citocinas (SLC). Como consecuencia, el paciente necesita ser hospitalizado durante la terapia. Además, el coste de manufactura y terapia es elevado, siendo una tecnología limitada a un sector muy bajo de la población. En este trabajo, mencionamos el empleo de una terapia emergente de células asesinas naturales (NK) con el receptor CAR (CAR-NK), que cuentan con muchas ventajas por encima de las células CAR-T. Las células CAR-NK conservan su capacidad citotóxica en contra de tumores gracias a su acción dependiente de receptores activadores e inhibidores, por lo que el receptor CAR, solo estimula sus habilidades y persistencia. Sumado a esto, el coste de una terapia de células CAR-NK podría resultar redituable debido a la capacidad de las células CAR-NK de eliminar múltiples células tumorales sin generar daño colateral en el paciente. Aquí analizamos las características de los múltiples receptores CAR y los fenotipos de células NK que han sido utilizados durante múltiples ensayos (NK-92, células NK de sangre cordal y periférica, y células NK iPSC).

Abstract One of the novel and effective devices against leukemia and solid tumors in adoptive immunotherapies is the use of the chimeric antigen receptor "CAR". CAR technology has been widely used in T-cells (CAR-T cells) empowering its efficacy on the identification and elimination of tumor cells, getting today certain drugs based on this technology. Nevertheless, CAR-T cells can have a negative impact on patient health, causing in many cases immune effector cell-associated neurotoxicity syndrome (ICANS) and cytokine release syndrome (CRS). As a consequence, the patient will have to be hospitalized for the duration of therapy. Moreover, the cost of manufacture and therapy is quite expensive, limiting its use to a low range of people. On the other hand, we analyze the advantages of Natural Killer cells with the CAR receptor (CAR-NK), which have many plusses over CAR-T cells. CAR-NK cells retain their cytotoxic abilities against tumor cells due their activator/ inhibitor receptors balance. Thus, the CAR receptor technology just increases their skills and persistence. Furthermore, CAR-NK therapy could be more profitable since CAR-NK can eliminate multiple tumor cells without generating collateral damage on patient health. Here, we discuss the characteristics of the multiples CAR receptors in general and the NK types cells that have been used in trials demonstrating their viable emerging therapy (NK-92, cord and peripheral blood NK cells, and iPSC-derived NK cells).

Effects of Bacillus firmus e volumine ex muris cellulae 6x on cytolytic activity of natural killer cells in vitro

Natural killer (NK) cells are among the first in defense of the innate immune system by eliminating a variety of abnormal or stressed cells such as cancer cells or virus-infected cells. Individuals who exhibit low cytolytic NK cell activity are believed to be at higher risk of viral infection, tumorigenesis, and various other diseases of the immune system. Therefore, restoration of impaired NK cell function might be an essential step in immunostimulatory therapy of immunocompromised patients. Bacillus firmus is a non-pathogenic gram-positive bacterium of the environment, which possesses various immunomodulatory properties in vitro and in vivo. This retrospective study reports on the effect of B. firmus on the activity of NK cells in vitro. Basal cytolytic NK cell activity against tumor cells among peripheral blood mononuclear cells (PBMCs) of routine patients was determined in a standardized NK cell cytotoxicity assay. The impact of cultivation of PBMCs with B. firmus preparation Bacillus firmus e volumine ex muris cellulae (Bacillus firmus (evc)) 6x on tumor cell killing by NK cells was monitored in relation to basal NK cell activity. This study showed that stimulation of PBMCs with Bacillus firmus (evc) 6x in vitro led to a significant increase in NK cell function. Substantial improvement in cytolytic NK cell activity (more than 1.3-fold of basal activity) was much more pronounced for patients with compromised NK cell function. Due to its immunostimulatory mode of action, Bacillus firmus (evc) may be of particular importance in therapy of patients with NK cell deficiency.

Sesamum indicum (sesame) enhances NK anti-cancer activity, modulates Th1/Th2 balance, and suppresses macrophage inflammatory response

Objective: To evaluate the potential immunomodulatory effects of an aqueous extract of Sesamum indicum seeds with regard to splenocyte proliferation, Th1/Th2 balance, macrophage function, and the cytotoxic activity of natural killer (NK) cells. Methods: Splenocyte proliferation was measu red by [3H]-thymidine incorporation. Griess assay was performed to evaluate the production of nitric oxide by macrophages. The levels of cytokines secreted by splenocytes and macrophages were measured by ELISA. JAM assay was performed to examine the cytotoxic activity of NK cells againstYAC-1 tumor cells. Results: Sesamum indicum significantly enhanced splenocyte proliferation in a dose-dependent manner. Sesamum indicum also increased and suppressed the secretion of Th1 and Th2 cytokines, respectively, by splenocytes. The secretion of key pro-inflammatory mediators (IL-6, TNFα, and nitric oxide) by primary macrophages was significantly inhibited by Sesamum indicum. Moreover, Sesamum indicum increased the cytotoxic activity of NK cells againstYAC-1 tumor cells. Conclusions: Sesamum indicum shows potent immunomodulatory, anti-inflammatory, and anti-cancer effects. Constituents of Sesamum indicum may be used as effective therapeutic agents in regulating immune reactions implicated in various infectious and non-infectious conditions including cancer.

Interferon-induced Transmembrane Protein 3 Prevents Acute Influenza Pathogenesis in Mice / 生物医学与环境科学（英文）

Objective@#Interferon-induced transmembrane protein 3 (IFITM3) is an important member of the IFITM family. However, the molecular mechanisms underlying its antiviral action have not been completely elucidated. Recent studies on IFITM3, particularly those focused on innate antiviral defense mechanisms, have shown that IFITM3 affects the body's adaptive immune response. The aim of this study was to determine the contribution of IFITM3 proteins to immune control of influenza infection .@*Methods@#We performed proteomics, flow cytometry, and immunohistochemistry analysis and used bioinformatics tools to systematically compare and analyze the differences in natural killer (NK) cell numbers, their activation, and their immune function in the lungs of -/- and wild-type mice.@*Results@#-/- mice developed more severe inflammation and apoptotic responses compared to wild-type mice. Moreover, the NK cell activation was higher in the lungs of -/- mice during acute influenza infection.@*Conclusions@#Based on our results, we speculate that the NK cells are more readily activated in the absence of IFITM3, increasing mortality in -/- mice.